利用CRISPR/Cas9腺病毒载体在鸡胚中进行基因敲入研究

为研究CRISPR/Cas9腺病毒载体在鸡胚中进行基因敲入的可行性,将包装不同滴度增强型绿色荧光报道基因(Enhance green fluorescent protein,EGFP)的腺病毒载体和慢病毒载体显微注射到HH14时期鸡胚的外周血管中,对胚胎发育至3.5 d和9d鸡胚存活、各器官中EGFP荧光强度等指标进行...     

The Effects of Ovine Lentivirus Infection on Some Productive Aspects in a Sardinian Sheep Flock from Italy

The effects of ovine lentivirus infection on the fat, protein and lactose concentrations on the somatic cell counts in ewes' milk, on milk production and on the birth weight and growth of lambs were studied in a flock of Sardinian sheep from central Italy. Data on 61 lactations of ewes positive to both the agar-gel immunodiffusion test and an enzyme-linked immunosorbent assay were compared with those on 46 lactations of seronegative ewes. Confounders such as parity, age, lactation length, litter size, and the sex of the lambs were considered. None of these traits seemed to be negatively influenced by the infection     

转录靶向猪Jiv基因shRNA细胞株的建立 及抗猪瘟病毒转基因猪的构建

【目的】构建转入靶向猪Jiv基因shRNA干扰片段的阳性细胞株,通过比较各细胞株对猪瘟病毒增殖的干扰效果,筛选对猪瘟病毒增殖有明显抑制作用的细胞株,为抗猪瘟转基因猪的构建提供材料。【方法】研究设计了靶向猪Jiv基因的4个shRNA干扰片段,并构建插入干扰片段的慢病毒(P1、P2、P3、P4)。将慢病毒分别转染PK-15细胞,阳性细胞接种猪瘟病毒后72 h用实时荧光定量PCR检测猪瘟病毒RNA的量,以比较4种干扰细胞株对猪瘟病毒增殖的干扰效果。将对猪瘟病毒增殖有较好干扰效果的P2慢病毒干扰载体转染猪胎儿成纤维细胞,获得稳定表达靶向猪Jiv基因shRNA干扰片段的猪胎儿成纤维细胞株,作为抗猪瘟病毒转基因猪构建的供体细胞,将细胞核移植到成熟的去核猪卵母细胞中,获得体细胞核移植胚胎,移植入受体母猪输卵管中进行转基因猪构建,对获得的转基因猪进行外源基因的鉴定。【结果】获得了4株转入靶向猪Jiv基因shRNA干扰片段的PK-15细胞株,其中转入P2干扰载体的细胞株对猪瘟病毒的增殖有明显的抑制作用,将转入P2干扰载体的猪胎儿成纤维细胞株为核供体细胞通过体细胞核移植,获得经鉴定为外源基因插入阳性的转基因猪。【结论】细胞Jiv基因的表达对猪瘟病毒的增殖有一定的影响,筛选获得的一个干扰细胞株对猪瘟病毒的增殖有明显的干扰效果;通过体细胞核移植技术获得一头转入靶向猪Jiv基因shRNA干扰片段（P2）的转基因猪。     

精子与慢病毒共孵育条件的优化以及转基因猪的制备

【目的】探索慢病毒与精子共孵育的方法,并制备转基因猪。【方法】采用正交试验分析精子密度、病毒量、精子与慢病毒共孵育时间、精子与慢病毒共孵育温度对精子活力的影响,采用PCR和Southern blotting检测转基因。【结果】①优化慢病毒与精子共孵育条件为：100 µL慢病毒原液（5×105cfu）与1.0×107个/mL精子在17℃下孵育30 min;②与病毒孵育后的精子（109个/mL）给母猪人工授精时,经PCR检测49头仔猪中有14头呈阳性;③Southern blotting结果表明,8头PCR阳性仔猪中,4头融合了外源基因。【结论】优化了精子与慢病毒共孵育的条件（100 µL慢病毒原液（5×105cfu）与1.0×107个/mL精子在17℃下孵育30 min）,成功制备了转基因猪,并检测有外源基因的融合。     

Construction of GDF9 Lentiviral Vector and Its Stable Expression in Goat Primary Fibroblasts

This study was aimed to constract growth differentiation factor 9 (GDF9) lentiviral vector,which was stably expressed in goat primary fibroblast. The CDS of sheep GDF9 gene was cloned by gene synthesis, the CDS was 1 362 bp, encoding 453 amino acids. After double enzyme digestion and ligation, the GDF9 fragment was sub-cloned into an empty lentiviral vector. The GDF9 lentiviral vector and the packaging plasmids were co-transfected into 293T cells, and the lentivirus with titer of 1×10⁶ TU/mL was obtained. The goat primary fibroblasts were transfected with GDF9 lentivirus, the red fluorescence could be observed in more than 60% of the cells, suggesting the prepared lentivirus had high infection efficiency to goat primary fibroblasts. After puromycin screening, all cells were able to observe red fluorescence. Real-time quantitative PCR analysis showed that the GDF9 expression level was higher than that of control group. The results indicated that the goat primary fibroblasts stably expressing GDF9 were successfully obtained. This results might lay a foundation for the function study of GDF9 and the goat germplasm resources innovation in the future.     

稳定表达猪瘟病毒E2蛋白的BHK细胞系的构建

猪瘟病毒(CSFV)的E2蛋白是引起猪体产生针对猪瘟保护性抗体的主要抗原,构建可稳定表达CSFV E2蛋白的细胞系,可为E2蛋白功能研究及基因工程猪瘟疫苗的研制提供物质基础。本研究将CSFV E2基因克隆至慢病毒表达载体,构建重组慢病毒表达质粒pCDH-E2。将pCDH-E2重组质粒与慢病毒包装质粒共转染293T细胞,获得重组慢病毒。将该慢病毒感染BHK-21细胞,经嘌呤霉素抗性筛选结合有限稀释法筛选出可表达CSFV E2蛋白的BHK细胞系。Western blot分析结果显示,所构建的重组细胞系传至第10代仍能稳定表达CSFV E2蛋白。该细胞系的建立为研制猪瘟新型重组疫苗及其生产奠定基础。     

自然感染的新疆美利奴羊中绵羊慢病毒的分离鉴定

本文从自然感染绵羊慢病毒的绵羊的外周白细胞及组织中分离病毒，在接种后２代均出现细胞病变效应，将病毒培养液浓缩１００倍作为抗原进行琼扩检测呈阳性反应，病毒培养液经免疫电镜检测，观察到了病毒颗粒。病毒的半数感染量约为１０－５／ｍｌ，将分离到的３株ＯＰＰＶ分别命名为ＮＸＪ－５５，ＮＸＪ－７３，ＮＸＪ－０４８１。     

Development of specific diagnostic test for small ruminant lentivirus genotype E

Small ruminant lentivirus (SRLV) belonging to the highly divergent genotype E has recently been identified in the Italian goat breed Roccaverano. In this report we have developed a specific serological test based on recombinant matrix/capsid antigen fusion protein. Performance has been evaluated and compared with a similar test based on genotype B antigen. Herds under study were selected according to the infectious status characterized by blood PCR and sequencing. Results clearly showed that B and E based recombinant ELISA only detected homologous infection and an apparent cross-reactivity was recorded in a herd in which co-infection was present. Three commercially available ELISAs showed different abilities in detecting genotype E infection, being the whole virus-based immunoassay the best choice. Genotype E-recombinant antigen was not detected in ELISA by three commercially available Mabs known to be cross-reactive among CAEV and MVV capsid antigens, further supporting the high divergence of the E genotype from others. Finally, a SRLV-free herd according to commercial ELISA testing, was analysed in the same area where genotype E was identified and few animals belonging to Roccaverano breed were found slightly reactive with the E antigens. Our results suggest that the prevalence of genotype E in other small ruminant populations may be conveniently estimated using a comparative assay based on a combination of genotype specific recombinant antigens and may highlight a wider space in which SRLVs evolve.     

Association between feline immunodeficiency virus antibodies and host characteristics in Finnish cats.

Toward the end of 1989 the largest private veterinary laboratory in Finland (Vet/lab) began using a commercial combined ELISA test for Feline Immunodeficiency Virus (FIV) antibodies and Feline Leukemia Virus (FeLV) antigens (Cite Combo). The overall proportion of FIV seropositive feline samples was 5% during the 22 month study period. The number of tests performed increased slowly while the positive test results decreased with time (7% in 1990 and 4% in 1991). The decrease in prevalence was assumed to reflect a change in the sample population rather than an actual change in the general cat population. There were more symptomatic and domestic cats tested in 1990 than 1991. The lower-risk groups in the second year of the study may simply be an indication that the cat owners became more aware of FIV and the motivation to send samples switched from the veterinarian's interest to diagnose the disease in a symptomatic cat to the owner's interest to survey their cats for possible FIV infection. In a multivariable analysis, breed, symptoms, age and sex were associated with the risk of FIV seropositivity. The risk increased faster with age in males than in females (i.e., the age effect was not constant between sexes). The cats with symptoms had a higher risk than those without symptoms and non-purebred cats were at a higher risk than purebred cats. FeLV infection was not associated with FIV.     

Ovine Lentivirus is Aetiologically Associated with Chronic Respiratory Disease of Sheep on the Laikipia Plateau in Kenya

A study was undertaken to investigate the occurrence of ovine lentivirus (OvLV) infection in sheep with chronic respiratory disease on the Laikipia Plateau, Kenya. All seven Merino crossbred sheep with chronic dyspnoea and emaciation examined for gross and microscopic lesions had lymphoid interstitial pneumonia (LIP), and one also had pulmonary abscesses. Two of the sheep with LIP also had lesions of ovine pulmonary carcinoma (OPC, jaagsiekte). Using in situ hybridization, OvLV DNA localized to a high proportion of pulmonary macrophages in lungs with lesions of LIP. Lung tissue samples from six of these sheep were positive for a syncytium-inducing virus in cultures of lamb testis cells. Thin-section electron microscopy of infected cells showed virions with morphogenesis typical of lentiviruses. In a western blotting assay, monoclonal antibodies to the OvLV capsid (CA, p27) and matrix (MA, p15) proteins of a North American OvLV isolate reacted with similar-sized bands of the virus, and serum from six of the sheep were reactive with CA from the Kenyan viral isolate. Using an OvLV agar gel immunodiffusion (AGID) test, all seven sheep were positive for serum antiviral antibody, as were 29% of 63 clinically normal sheep from Laikipia District. However, when sera from the healthy sheep were tested in a western blot assay, only 52% had IgG reactive to the OvLV CA, indicating a high rate of false negative reactions with the AGID test. Serum samples from 87 Red Maasai or Dorper crossbred sheep from two farms in other parts of Kenya were OvLV seronegative by both the AGID test and the western blot assay. These results document the first identification of OvLV as a cause of chronic respiratory disease in sheep in Kenya and show a high rate of infection in sheep flocks, with a high prevalence of chronic respiratory disease.